MicroRNA (miRNA) has become an ideal biomarker candidate for cancer diagnosis, prognosis, and therapy. In this study, we have developed a novel one-step method for sensitive and specific miRNA detection via enzymatic signal amplification and demonstrated its practical application in biological samples. The proposed signal amplification strategy is an integrated "biological circuit" designed to initiate a cascade of enzymatic polymerization reactions in order to detect, amplify, and measure a specific miRNA sequence by using the isothermal strand-displacement property of a mesophilic DNA polymerase together with the nicking activity of a restriction endonuclease. The circuit is composed of two molecular switches operating in series: the nicking endonuclease-assisted isothermal polymerization reaction activated by a specific miRNA and the strand-displacement polymerization reaction designed to initiate molecular beacon-assisted amplification and signal transduction. The hsa-miR-141 (miR-141) was chosen as a target miRNA because its level specifically elevates in prostate cancer. The proposed method allowed quantitative sequence-specific detection of miR-141 in a dynamic range from 1 fM to 100 nM, with an excellent ability to discriminate differences among miR-200 family members. Moreover, the detection assay was applied to quantify miR-141 in cancerous cell lysates. The results are in excellent agreement with those from the reverse transcription polymerase chain reaction method. On the basis of these findings, we believe that this proposed sensitive and specific assay has great potential as a miRNA quantification method for use in biomedical research and clinical diagnosis.